Each year 700,000 US patients require administration of a rapid onset anticoagulant (ROA) to facilitate cardiac surgical procedures that involve cardiopulmonary bypass (CPB). Unfractionated heparin (UFH) is the standard ROA used for CPB but it presents major toxicities, including serious drug-induced bleeding, that have prompted efforts to identify ROA alternatives that are safe yet effective. RNA aptamers that inhibit specific clotting factors are an emerging class of therapeutic anticoagulants. Because these oligonucleotides can be rapidly reversed using sequence complementary antidotes, RNA aptamers offer an improved safety profile by minimizing risks of drug-associated bleeding. Our lab has developed several anticoagulant aptamer/antidote pairs that target distinct clotting factor enzymes, including ones that have shown therapeutic efficacy in non- CPB clinical settings. Unfortunately, in comparison to UFH, individual aptamers cannot provide sufficient anticoagulation for highly thrombogenic procedures like CPB, and thus further research is needed to enhance aptamer-based ROAs for this important indication. Factor (F)Xa is a key coagulation enzyme that, upon assembly with its cofactor FVa to form the prothrombinase complex, catalyzes the production of thrombin which directly triggers clot formation. In this light I helped to discover that the reversible anticoagulant intensity of our FXa aptamer, 11F7t, can be synergistically potentiated by adding a clinical inhibitor that selectively impedes FXa's catalytic site, to thereby reproduce the in vitro anticoagulant effect of UFH. In addition, such combinations appear to be more effective than UFH in reducing thrombin generation, which is linked with several causes of morbidity post-CPB. Hence, in this proposal, I aim to (1) investigate the potential utility of a novel ROA strategy for CPB that employs synergizing combinations of 11F7t and a FXa catalytic site inhibitor, and (2) study the mechanism of this anticoagulant synergism in a manner that will not only help elucidate the molecular processes that govern normal blood clotting, but also facilitate the design of future ROA therapies. 11F7t does not affect FXa's catalytic activity but instead seems to act by binding an unknown exosite on FXa that disrupts prothrombinase formation. Thus, I propose structural studies to identify the functional exosite on FXa that mediates 11F7t binding, and enzyme kinetics studies to interrogate the mechanism of anticoagulant synergy between 11F7t and a FXa catalytic site inhibitor. Moreover, I intend to evaluate the efficacy of this innovative ROA strategy in models of ex vivo human extracorporeal membrane oxygenation (ECMO) and in vivo porcine CPB. Successful completion of this proposal will enhance basic understanding of mechanisms that regulate hemostasis, and may also identify synergizing FXa aptamer/catalytic site inhibitor combinations that can yield effective ROA substitutes for UFH to improve patient outcomes after CPB.